1. Field of the Invention
The present invention relates to a reaction chip and a reaction method suitably used for a biochemical reaction such as a chemical reaction, DNA reaction, and protein reaction and a temperature controlling unit and a gene treating apparatus including the temperature controlling unit for treatment such as amplification on genes contained in a biological sample.
2. Description of the Related Art
In recent years, in the field of, for example, a biochemical reaction such as a chemical reaction, DNA reaction, and protein reaction, a technology called a μ-TAS (Total Analysis System) or Lab-on-Chip is studied and put to practical use as a technique to treat a very small quantity of sample solution on a chip. This enables a reaction experiment, which required large-scale laboratory equipment and a large quantity of reaction reagents in the past, to perform with a small quantity of reaction reagents using a reaction chip measuring several mm or less pre side.
Examples of this kind of biochemical reaction include a DNA amplification reaction by an enzyme reaction, hybridization reaction to detect a sequence of specimen DNA by using a probe DNA having a known sequence, and detection reaction of SNP (monobasic polymorphism) in a DNA sequence. The invader (registered trademark) method and the TaqMan PCR method are known as SNP detection methods (see, for example, Patent Document 1).
When these reactions are caused using a chip, for example, to decide the sequence of a gene or DNA, a method by which a probe DNA is fixed onto slide glass to allow a hybridization reaction thereon is known.
Further, a method by which a microscopic hole or dent called a well is formed on a chip to use the well as a reaction field is known.
A plurality of well-shaped reaction containers is mutually connected by a reagent solution channel installed from a reagent reservoir part (see, for example, Patent Document 2). When a reagent solution is fed by using such a channel, it is important to fill a reaction container with the reagent solution to prevent bubbles from being left behind. If bubbles remain inside the reaction container, quantities and concentrations of the reagent solution in each reaction container fluctuate, leading to fluctuations of reaction states. Moreover, even if reaction states do not fluctuate, it is extremely probable that an error of photometric intensity is caused by the bubbles.
Thus, several methods are proposed to remove bubbles from the reaction container.
As a method thereof, a liquid circuit having at least one channel inside a layered product formed by laminating a plurality of substrates in which a communication hole communicating the channel and outside by passing through at least one substrate is proposed (see, for example, Patent Document 3). Patent Document 3 discloses that the communication hole is a hole formed in a single-crystal silicon substrate or glass substrate by using photolithography and has a tapered inner circumferential surface with an increasingly smaller opening area from the channel toward the outside to remove bubbles to the outside through the communication hole. Further, the communication hole preferably has at least hydrophobicity and Patent Document 3 describes for this purpose that the substrate itself has hydrophobicity or adding hydrophobicity to a substrate having no hydrophobicity afterwards.
Further, a reaction chip having a channel passing through a first surface and a second surface and including a bubble trap to separate bubbles fed together with a sample in each sample hole is proposed (see, for example, Patent Document 4).
Incidentally, a reagent solution fed to a chip used for reaction analysis is frequently a reagent solution having high viscosity such as organic substance for a chemical reaction and extracted DNA, synthetic DNA, and enzyme for a biochemical reaction. When using such a reagent solution, according to the method described in Patent Document 2 or 3, there is a possibility that bubbles are not sufficiently removed or separated so that bubbles remain in the reaction container. Moreover, the method described in Patent Document 2 or 3 is a technology applicable only to a so-called open reaction container that is open to the outer space and is not applicable to a closed reaction container whose outer circumference is completely enclosed by walls.
In addition, a method of making a reaction container hydrophobic with surface treatment such as corona treatment and plasma treatment is frequently used to remove bubbles. In this case, however, surface modification of the reaction container may occur to result in different batch reaction conditions such as a change in pH, posing a problem of a possibility of an intended desired reaction from being blocked. Moreover, if plasma treatment is applied, surface modification of the reaction container occurs, but the surface modification is hard to persist so that there is a problem that the state immediately after treatment cannot be maintained.
When a reaction is caused using these analysis chips, a reaction reagent is first arranged inside a plurality of well-shaped reaction containers. Next, a reaction reagent solution is fed to the plurality of well-shaped reaction containers via a channel by infusing the reaction reagent solution into the analysis chip. Accordingly, the fixing reagent and the reaction reagent solution come into contact to start a reaction. The well-shaped reaction containers are heated during reaction if necessary.
However, according to the above reaction method, when the reaction reagent solution is fed into the well-shaped reaction container via the channel, there is a possibility that the fixing reagent prearranged inside the well-shaped reaction container flows out to adjacent well-shaped reaction containers. Accordingly, there is a problem that contamination may be caused. There is also a possibility that the fixing reagent, reaction reagent solution, or fluorescent substance for detection is diffused into the adjacent well-shaped reaction containers during reaction in each well-shaped reaction container. Accordingly, there is a problem that it becomes impossible to measure accurate reaction data.
Thus, a reaction chip and a reaction method capable of preventing an occurrence of such contamination and measuring accurate reaction data are disclosed (see, for example, Patent Document 5).
The reaction chip described in Patent Document 5 is constituted by a substrate forming a well-shaped reaction container and a cover material covering the substrate. The reaction method using the reaction chip is to cover a reaction reagent arranged in the well-shaped reaction container with a hot-melt sealing compound and feed the reaction reagent solution on top of the sealing compound before the sealing compound being melted by heating to bring the reaction reagent and the reaction reagent solution into contact. According to the method, the reaction reagent will not flow out to adjacent well-shaped reaction containers, so that contamination can be prevented from occurring.
For a reaction chip used for biochemical reaction such as an enzyme reaction, it is advantageous to use a substrate with a high thermal conductivity because such a reaction frequently requires heating a reagent. However, if a reaction reagent is fixed onto a substrate with a high thermal conductivity, heat when the substrate and cover material were stuck together may be conducted to the reaction reagent, posing a problem that activity of the reaction reagent is lowered or devitalized. Speaking of, for example, the reaction method described in Patent Document 5, it is desirable to use a substrate with a high thermal conductivity as a substrate on the side on which a well-shaped reaction container is formed, but in such a case, the above problem is caused.
If a reaction reagent is fixed onto a substrate with a high thermal conductivity and the reaction reagent is covered, like the method in Patent Document 5, with a hot-melt sealing compound, the sealing compound is melted by heat when the substrate and cover material were stuck together, posing a problem that the sealing compound flows out to a channel of the reaction chip to block the channel or the shape of the sealing compound when re-solidified becomes unstable, leading to incomplete sealing of the reaction reagent. Because of this problem, there is a possibility that contamination cannot be sufficiently prevented from occurring.
In these genetic tests, the amount of nucleic acid (DNA) contained in a sample is amplified by the polymerase chain reaction (PCR) for the test and an attempt is being made to make the test faster by reducing the time necessary for the PCR.
As a method of executing the PCR in a shorter time, an attempt is being made to execute the PCR with a smaller amount of sample and a reaction container and a reaction apparatus (temperature controlling unit) therefor are devised.
Most reaction containers are made of synthetic resin that does not inhibit a biological reaction and a reaction is caused by reducing a reaction volume to several tens microliter. In addition, in some instances, the reaction container is formed from aluminum.
In such reaction containers, a PCR reaction is allowed to occur without a minimum amount of sample being evaporated by a heating unit being brought into contact from above and below by a reaction apparatus described in Patent Document 6 or 7.
The reaction apparatus described in Patent Document 6 or 7 causes no big problem if the reaction container is formed from a single material. However, if a PCR reaction is allowed to occur by using a container constructed by separate materials having different thermal conductivities in an upper part and a lower part of the reaction container for the purpose of improving performance of the reaction container, the temperature distribution of the sample inside the reaction container becomes inhomogeneous due to a difference in thermal conductivity, posing a problem that the PCR reaction does not proceed smoothly.    Patent Document 1: Japanese Patent Application Laid-Open No. 2002-300894    Patent Document 2: Japanese Patent Application National Publication No. 2002-503336    Patent Document 3: Japanese Patent Application Laid-Open No. 9-257748    Patent Document 4: Japanese Patent No. 2955229    Patent Document 5: Japanese Patent Application Laid-Open No. 2007-090290    Patent Document 6: Japanese Patent No. 3661112    Patent Document 7: Japanese Patent No. 3686917